CN102448987B - Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody - Google Patents

Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody Download PDF

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CN102448987B
CN102448987B CN201080018409.6A CN201080018409A CN102448987B CN 102448987 B CN102448987 B CN 102448987B CN 201080018409 A CN201080018409 A CN 201080018409A CN 102448987 B CN102448987 B CN 102448987B
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李森伟
柯耀煌
张永克
朱伟民
余国良
马梵辛
樊馨
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Jiangsu Xiansheng Biopharmaceutical Co ltd
Apexigen Inc
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Epitomics Inc
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Abstract

The present invention provides an anti-VEGF monoclonal antibody, which has the variable region of heavy chain comprising the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and/or the variable region of light chain comprising the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The antibody can be produced by the cell line with the preservation number of CGMCC NO.3233. The invention also provides the use of said antibody for the manufacture of medicaments for the treatment of a disease that is relevant to VEGF, wherein further provided with pharmaceutical composition, agents, kits and chips comprising said antibody, as well as the cell line with the preservation number of CGMCC NO.3233.

Description

The monoclonal antibody of a kind of anti-VEGF and the pharmaceutical composition that contains this antibody
The application requires to submit on August 28th, 2009 right of priority of the Chinese patent application that Patent Office of the People's Republic of China, application number are 200910171550.9, denomination of invention is " monoclonal antibody of a kind of anti-VEGF and the pharmaceutical composition that contains this antibody ", and its full content is by reference in conjunction with in this application.
Technical field
The present invention relates to genetic engineering antibody technical field, be specifically related to genetic engineering antibody and the pharmaceutical composition that contains this antibody and test kit with vascular endothelial growth factor VEGF specific binding.
Background technology
Vascular endothelial growth factor, vascular endothelial growth factor, being called for short VEGF, is the specific heparin binding growth factor of vascular endothelial cell (heparin-binding growth factor), induction of vascular new life in vivo.People's vegf protein is in the scientist by the U.S. in 1989 success purifying and qualification, and clone and measured its gene order.
Vascular endothelial growth factor has the effect that promotes vasculogenesis.The member of all VEGF family can carry out activating cells reaction by the corresponding acceptor (VEGFRs) in conjunction with cell surface, thereby by phosphatizing dimerization activation.The ectodomain that vascular endothelial growth factor receptor contains 7 immunoglobulin-likes, a membrane spaning domain and a born of the same parents' intracellular domain containing tyrosine kinase domain.VEGF-A can with vascular endothelial growth factor receptor 1 (receptor Flt-1-1) and vascular endothelial growth factor receptor-2 (KDR/Flk-1) combination.Vascular endothelial growth factor receptor-2 have almost mediated all known cell responses of VEGF.Vascular endothelial growth factor, its biological activity and its acceptor are elaborated and study by people such as people and Marti such as Matsumoto (referring to Angiogenesis in ischemic disease.Thromb Haemost.1999Suppl 1:44-52; VEGF receptor signal transduction Sci STKE.2001:RE21).
VEGF is the homodimer glycoprotein of high conservative, and the strand that is respectively 24kDa by two molecular weight forms dimer with disulfide linkage.Due to the different cut mode of mRNA, produce respectively at least 5 kinds of albumen forms such as VEGF121, VEGF145, VEGF165, VEGF185, VEGF206, wherein VEGF121, VEGF145, VEGF165 are secretor type soluble proteinss, can directly act on vascular endothelial cell, promote vascular endothelial cell proliferation and migration, increase vascular permeability.VEGF relative disease is common to be characterized as: propagation, vascular permeability increase, tissue edema and the inflammation of excessive vascular endothelial cell are as the cerebral edema being caused by damage, apoplexy or tumour; The oedema that inflammatory disease causes, as psoriatic or sacroiliitis, comprises rheumatoid arthritis; Asthma; The relevant anasarca of burning; The ascites that tumour, inflammation or wound cause and hydrothorax; Chronic tracheitis; Capillary leak syndromes; Septicemia; The kidney disease that proteinaceous effluent is relevant; Eye disease, as senile macular degeneration SMD and diabetic retinopathy; Tumour, comprises mammary cancer, lung cancer, colorectal cancer, cerebral glioma and kidney etc.
The combination of antibody and its target spot is specific, can play the effect of mediated immunity effect mechanism, and in serum, has the longer transformation period.These characteristics make antibody have very strong treatment application.
At present, FDA and European approved recombinant humanized mouse-anti VEGF monoclonal antibody AVASTIN be used for the treatment of colorectal cancer, nonsmall-cell lung cancer, mammary cancer, cerebral glioma, kidney and senile macular degeneration SMD (AMD), the sales volume of 2008 reaches 4,800,000,000 dollars.But AVASTIN antibody is not high to VEGF avidity, in addition owing to producing without competition, the expense of client need cost great number, the previous patient's medication of the order expense of a year is about 50,000 to 100,000 dollars.So, research and develop new anti-VEGF monoclonal antibody, thereby reduce patient's burden, reducing medical expense is a problem demanding prompt solution.
Definition
Before further setting forth the present invention, we are necessary to recognize, the present invention is not limited to the specific embodiment of description, that is to say, on specific form, may exist variation.Also have and a bit need to remind, because scope of the present invention is subject to the restriction of additional claims, therefore, term used herein is just in order to describe the object of particular, instead of in order to limit object of the present invention.
Term " antibody " and " immunoglobulin (Ig) " can exchange use in this article.The term that these terms are well known to the skilled person, specifically refers to the protein being made up of one or more polypeptide of energy specific combination antigen.A kind of form of antibody has formed the basic structural unit of antibody.This form is tetramer, and it is made up of two pairs of identical antibody chains, every a pair of have a light chain and a heavy chain.In every antagonist chain, the variable region gang of light chain and heavy chain is responsible for conjugated antigen jointly, and constant region is responsible for the effector functions of antibody.
Known immunoglobulin polypeptides comprises κ and lambda light chain at present, and alpha, gamma (IgG 1, IgG 2, IgG 3, IgG 4), δ, ε and μ heavy chain or their other type equivalence thing.The immunoglobulin (Ig) " light chain " (approximately 25kDa or about 214 amino acid) of total length comprises one by NH 2about 110 amino acids formed variable regions on-end, and κ or λ constant region on a COOH-end.The immunoglobulin (Ig) " heavy chain " (approximately 50kDa or about 446 amino acid) of total length, comprise equally a variable region (about 116 amino acid), one of and CH, for example γ (about 330 amino acid).
Term " antibody " and " immunoglobulin (Ig) " comprise antibody or the immunoglobulin (Ig) of any phenogen, or the maintenance antibody fragment of being combined with antigen-specific, include but not limited to Fab, Fv, the fused protein of scFv and Fd fragment, chimeric antibody, humanized antibody, single-chain antibody and the antigen-binding portion thereof that comprises antibody and non-antibody protein.Antibody can be labeled and detect, for example, and can be by radio isotope, can produce the enzyme, fluorescence protein, vitamin H that can detect thing etc. and carry out mark detected.Antibody can also be incorporated into solid phase carrier, includes but not limited to polystyrene plate or bead etc.This term also comprises Fab ', Fv, F (ab ') 2and/or other antibody fragment and the monoclonal antibody that can be combined with antigen-specific.
Antibody can also exist in a variety of forms, for example, comprise Fv, Fab and (Fab ') 2, and difunctional hybrid antibody (for example document, Lanzavecchia etc., Eur.J.Immunol., 1987; 17,105) and for example, with single stranded form (, Huston etc., Proc.Natl.Acad.Sci.U.S.A., 1988; 85,5879 and Bird etc., Science, 1988; 242,423, be incorporated herein by reference) exist.(form also referred to as " complementary determining region " or CDR), these hypervariable regions are by framework region (FR) interval by three hypervariable regions for the heavy chain of immunoglobulin (Ig) or variable region of light chain.The scope of framework region and complementary determining region by explication (referring to " Sequences of Proteins of Immunological Interest; " E.Kabat etc., U.S.Department of Health and Human Services, 1991).The sequence of all antibody aminoacid sequences that discuss in this place is all with reference to Kabat system.The different light chain of same species is relative conservative with heavy chain framework region sequence.The framework region of antibody is for location and calibration CDR.CDR is mainly responsible for the epi-position of conjugated antigen.
Chimeric antibody is its heavy chain and the antibody of light chain gene through building, and particularly utilizes the genetic engineering modified antibody variable region that belongs to different plant species and constant region gene.For example, the variable region fragment of mouse monoclonal antibody gene can be connected to people's antibody constant region fragment as γ 1 and γ 3.For example treatment is a kind of chimeric protein with chimeric antibody, its origin comes from rabbit antibody variable region fragment or antigen binding domain fragment and people's antibody constant region or effect district combination (as the anti-Tac chimeric antibody of being prepared by the cell of A.T.C.C. preservation registration number CRL 9688), certainly, the gene source of chimeric antibody also can use other mammalian species.
Term " humanized antibody " is identical with " Humanized immunoglobulin " implication, conventionally, compared with the non-humanization form of humanized antibody and same antibody, can be reduced in the immune response producing in human host.
Be appreciated that the humanized antibody that the present invention designs and produces may substitute some conservative amino acid, these amino acid there is no impact to antigen combination or other functions of antibody.In other words, gly and ala; Val, ile and leu; Asp and glu; Asn and gln; Ser and thr; Lys and arg; Phe and tyr, the inner amino acid of above each combination can replace mutually.The amino acid not being present in same group belongs to " substantially different " amino acid.
In certain embodiments, the avidity K between antibody and its target spot d(dissociation constant) characterizes, and it is lower than 10 -6m, 10 -7m, 10 -8m, 10 -9m, 10 -10m, 10 -11m or approximately 10 -12m or lower.
" variable region " of heavy chain of antibody or light chain is that the N of this chain holds ripe region.All Ranges, CDR and residue numbering are all taking sequence alignment, define as basis by existing structure knowledge.The qualification of framework region and CDR residue and numbering are pressed Chothia and (Chothia, Structural determinants in the sequences of immunoglobulin variable domain.J Mol Biol.1998 described in other people; 278,457).
VH is the variable region of heavy chain of antibody.VL is the variable region of light chain of antibody, and it may have κ and λ isotype.K-1 antibody has κ-1 isotype and K-2 antibody has κ-2 isotype, and V λ is variable lambda light chain.
Term " polypeptide " and " protein " can exchange use in this article, they all refer to the amino acid of the polymerized form of any length, can comprise coding and noncoding amino acid, by chemistry or biochemical modification or derivative amino acid and have the polypeptide of modified peptides skeleton.This term comprises fusion rotein, includes but not limited to have the fusion rotein of allogeneic amino acid sequence; There is allos and homology leader sequence, the fusion rotein of with or without N-end methionine residues; With the albumen of immune label; With the fusion rotein that can detect fusion partner, for example, comprise that fluorescence protein, beta-galactosidase enzymes, fluorescein etc. are as fusion rotein of fusion partner etc.Polypeptide can have any size, and term " peptide " refers to that length is the polypeptide of 8-50 residue (as 8-20 residue).
Term " experimenter ", " host ", " patient " and " individuality " can be used alternatingly in this article, specifically refer to accept any Mammals of diagnosis or treatment, refer in particular to the mankind.Other object may comprise monkey, ox, dog, cat, cavy, rabbit, rat, mouse and horse etc.
" corresponding amino acid ", refers in the time of two or more aminoacid sequence comparison, is positioned at the amino-acid residue of same position (namely they correspond to each other).The method of antibody sequence comparison and numbering is at Chothia, and on seeing, Kabat, is shown in above and in other and obtains elaboration.Those of ordinary skill in the art are known (referring to as Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), sometimes can in one or two amino acid of antibody, manufacture one, two or three breach and/or insert 1,2,3 or 4 residue or approximately 15 residues (particularly in L3 and H3 CDR) at the most, thereby complete once comparison.
" can the position of substitution ", refers to a specific position of antibody, and it can not be made by different aminoacid replacement, and the combination of antibody is active significantly to be reduced.Qualification can the position of substitution method and they can how be substituted in below and will be further described in more detail.Can also can be called " variation tolerance position " by the position of substitution.
" parent " antibody refers to the target antibody as aminoacid replacement.In certain embodiments, " donor " antibody can " be given tax " by amino acid to parental antibody to generate the antibody changing." associated antibodies " refers to the antibody that has similar sequences and produced by the cell with common B cell ancestors.This B cell ancestors are contained and are had the light chain VJC district of rearrangement and reset heavy chain VDJC district genome, and produce the antibody that does not also experience affinity maturation." purity " or " original " B cell being present in spleen tissue is the common ancestor of B cell.Associated antibodies is conventionally very similar in sequence with identical epitope combination, particularly their L3 and H3 CDR.The H3 of associated antibodies and L3 CDR have identical length and are close to consistent sequence (have 0-4 amino-acid residue different).Associated antibodies is by common antibody ancestors, and the antibody that originally B cell ancestors produce is associated.
Summary of the invention
The object of this invention is to provide a kind of monoclonal antibody higher to VEGF avidity.VEGF monoclonal antibody of the present invention, the aminoacid sequence shown in SEQ ID NO.1, SEQ ID N0.2 and SEQ ID NO.3 is contained in its variable region of heavy chain, and/or the aminoacid sequence shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 is contained in its variable region of light chain.
" antibody " of the present invention should be interpreted as containing any specific binding factor with required specific binding domains.Thereby function equivalent and the homologue of the antibody fragment of homology, derivative, humanized antibody and antibody with it contained in this term, also comprises any polypeptide that contains antigen binding domains, no matter be natural or synthetic generation.The example of antibody is immunoglobulin (Ig) hypotype (as IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Also can be comprise antigen binding domains fragment as Fab, scFv, Fv, dAb, Fd; And double-chain antibody (diabodies).Fusion to mosaic molecule or equivalent another polypeptide, that comprise antigen binding domains is also included within wherein.The cloning and expression of chimeric antibody is described in EP.A-0120694 and EP.A.0125023.
Monoclonal antibody of the present invention can be, for example, derivative, function equivalent and homologue unit price or single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, also comprise antibody fragment and any polypeptide that contains antigen binding domains.
Antibody can be modified by many modes, can produce other antibody or the chimeric molecule that retain original antibodies specific with DNA recombinant technology.This technology can comprise that constant region or constant region that the DNA of the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) is introduced to different immunoglobulin (Ig)s add framework region.Referring to, EP.A.184187, GB 2188638A or .EP.A.239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of produced antibody.
Hypervariable region CDR1, the CDR2 and CDR3 and catenation sequence of monoclonal antibody of the present invention in heavy chain and light chain, other is framework region.Framework region can be replaced by other sequences under impregnable condition in the three-dimensional structure in conjunction with required, and the molecular basis of antibodies specific mainly comes from its hypervariable region CDR1, CDR2 and CDR3, and these regions are the key positions of being combined with antigen.For maintaining preferred binding characteristic, the sequence of CDR should retain as far as possible, but, may need some amino acid changes to make binding characteristic optimization, those skilled in the art can reach this object with standing procedure.
In some preference, a variable region that monoclonal antibody comprises following content: has comprised as the variable region of heavy chain of SEQ ID NO.7, and it contains CDR1 (SNNDVMCW; SEQ ID NO.1), CDR2 (GCIMTTDVVTEYANWAKS; SEQ ID NO.2) and CDR3 (RDSVGSPLMSFDLW; SEQ ID NO.3); With one comprised as the variable region of light chain of SEQ ID NO.8, its CDR1 (QASQSIYNNNELS; SEQ ID NO.4), CDR2 (RASTLAS; SEQ ID NO.5), and CDR3 (GGYKSYSNDGNG; SEQ ID NO.6).
The variant of variable region CDR and above-mentioned CDR district are consistent (for example, 1,2,3,4 or 5 aminoacid replacement) substantially except having 6 aminoacid replacement at the most different, have to be combined activity with VEGF in this monoclonal antibody CDR district.
In other embodiments, antibody can comprise: an a) variable region of heavy chain, and its aminoacid sequence is compared from SEQ ID NO.7 the different of 6 aminoacid sequence replacements at the most, for example 1,2,3,4,5 or 6 replacement; And a b) variable region of light chain, its aminoacid sequence is compared have at the most 6 aminoacid sequences replace different, for example 1,2,3,4,5 or 6 aminoacid replacement from SEQ ID NO.8.Target antibody may comprise these any one or its combinations in replacing.
Have the antibody of any in these the position of substitution and there is equally the activity in conjunction with VEGF with the antibody that has all the position of substitution.Aminoacid replacement can be present in framework region HeCDR district simultaneously, or appears at separately framework region HuoCDR district.Therefore, in some preference, the aminoacid sequence of the framework region of variable region of heavy chain is compared may have maximum 6 aminoacid sequences to replace different from SEQ ID NO.7, for example 1,2,3,4,5 or 6 replacement, compare may have maximum 6 aminoacid sequences to replace different, for example 1,2,3,4,5 or 6 replacement from SEQ ID NO.8 with the aminoacid sequence of the framework region of variable region of light chain.
In some antibody, aminoacid replacement may be distributed in multiple CDR district.Therefore, the aminoacid sequence in multiple CDR district of variable region of heavy chain is compared from SEQ ID NO.7 may the different of 6 aminoacid sequence replacements at the most, for example 1,2,3,4,5 or 6 replacement, compare may have at the most 6 aminoacid sequences replace different, for example 1,2,3,4,5 or 6 replacement from SEQ ID NO.8 with the aminoacid sequence in multiple CDR district of variable region of light chain.
In special preference, antibody may comprise an a) variable region of heavy chain, its aminoacid sequence and the SEQ ID NO.7 mono-b) variable region of light chain of making peace, and its aminoacid sequence is consistent with SEQ IDNO.8.
In special preference, antibody may comprise an a) variable region of heavy chain, its aminoacid sequence and SEQ ID NO.7 have 95% consistence and a b) variable region of light chain at least, and its aminoacid sequence and SEQ ID NO.8 have 95% consistence at least.Therefore, target antibody may comprise an a) variable region of heavy chain, its aminoacid sequence and SEQ ID NO.7 at least have an appointment 95%, 96%, 97%, 98%, 99% or 100% consistence and a b) variable region of light chain, at least have an appointment 95%, 96%, 97%, 98%, 99% or 100% consistence of its aminoacid sequence and SEQ ID NO.8.
In special preference, antibody may comprise an a) heavy chain, its aminoacid sequence and the SEQID NO.9 mono-b) light chain of making peace, and its aminoacid sequence is consistent with SEQ ID NO.10.
In special preference, antibody may comprise an a) heavy chain, and its aminoacid sequence and SEQID NO.9 have 95% consistence and a b) light chain at least, and its aminoacid sequence and SEQ ID NO.10 have 95% consistence at least.Therefore, target antibody may comprise an a) heavy chain, its aminoacid sequence and SEQ ID NO.9 at least have an appointment 95%, 96%, 97%, 98%, 99% or 100% consistence and a b) light chain, at least have an appointment 95%, 96%, 97%, 98%, 99% or 100% consistence of its aminoacid sequence and SEQ ID NO.10.Except aminoacid replacement described above, target antibody may have additional amino acid at the two ends of heavy chain or light chain.For example, target antibody may comprise respectively at least 1,2,3,4,5 or 6 or more plus Amino Acid at the C of heavy chain and/or light chain or N-terminal.In certain embodiments, target antibody may be shorter than exemplary amino acid as described herein, and its key distinction is that two ends at heavy chain and light chain are respectively than few 1,2,3,4,5 or 6 amino acid of exemplary amino acid.
Target antibody may be humanized.Generally speaking, humanized antibody is to carry out aminoacid replacement by the framework region at parental antibody to produce the antibody of modification, and humanized antibody has less immunogenicity compared with parental antibody.Antibody can carry out humanization by a lot of technology well known in the art, comprises, for example, CDR transplants (EP.A-239,400; The open text WO 91/09967 of PCT; U.S.Pat.No.5,225,539; 5,530,101; With 5,585,089), and chain is replaced (U.S.Pat.No.5,565,332).In some preference, it is confirmed framework residue for the importance of antigen combination and confirmed the extraordinary framework residue of special site by sequence alignment by the interaction of simulation CDR and framework residue that framework is replaced.(see, for example, U.S.Pat.No.5,585,089; Riechmann etal., Nature, 1988; 332,323).The detail of the humanization method of antibody can, with reference to U.S. Patent application 10/984,473, be submitted on November 8th, 2004, and title is " Methods for antibody engineering ", and this application is all incorporated herein by reference.Generally speaking, this humanized method comprises by comparison can determine suitable site in conjunction with the sequence of the antibody of same antigen, and replaces the amino acid in this site with the different aminoacids that similar amino acid is positioned at same loci.In these methods, the antibody that the aminoacid sequence of parental antibody is relevant to other compares (for example, sequence alignment), thus identification variation tolerance position.The aminoacid sequence of the parental antibody variable region aminoacid sequence conventionally and in human antibodies database compares, and selects this and parental antibody and have the humanized antibody of similar aminoacid sequence.The sequence of parental antibody and humanized antibody is compared (for example, sequence alignment), in parental antibody, one or more variations tolerate locational amino acid and are replaced by the amino acid on corresponding position in human antibody.
The substitution technique of variation tolerance discussed above position is combined with any known humanization method easily, and be also applied to easily and produce the humanized antibody that comprises CDR district, this antibody CDR district modifies on the basis in faithful to parental antibody CDR district.Therefore, the present invention also provides and has comprised the humanization VEGF neutralizing antibody that changes multiple CDR district of version from parental antibody.
Than the dissociation constant Kd of prior art product A VASTIN and VEGF, low (monoclonal antibody Kd of the present invention is 0.485nM to the anti-VEGF monoclonal antibody in humanization rabbit source that the present invention discusses, AVASTIN is 47.9nM), the avidity of monoclonal antibody of the present invention and VEGF is higher, shows that the present invention has stronger restraining effect to VEGF.In mouse model test, show that antibody tumour inhibiting rate of the present invention is apparently higher than AVASTIN (being shown in embodiment 5), so potential clinical efficacy of the present invention will be higher than AVASTIN in theory.
In one embodiment, the cell strain that monoclonal antibody of the present invention is CGMCC No.3233 by deposit number produces, its heavy chain amino acid sequence is as shown in SEQ ID NO.9, light chain is as shown in the SEQ ID NO.10, itself and VEGF dissociation constant are 0.485nM, be 1/100 of AVASTIN, illustrate that the binding ability of EPI0030 and VEGF is better than AVASTIN.
The present invention also provides a kind of cell strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.3233.It produces a kind of monoclonal antibody, described heavy chain of antibody aminoacid sequence is as shown in SEQ ID NO.9, light chain is as shown in SEQ ID NO.10, in an embodiment of the present invention, called after EPI0030 antibody, test cell line and animal vivo test prove, can suppress in vitro endothelial cell proliferation and the migration of VEGF induction, and can suppress in animal body tumor growth, can be used for treating VEGF diseases related.
Monoclonal antibody described in the present invention also provides is in the purposes for the preparation of in the diseases related medicine for the treatment of VEGF.Diseases related tumour, senile macular degeneration SMD, nerve degenerative diseases, obesity, the diabetes of comprising of described VEGF.This target antibody can be for the scientific research relevant to VEGF, as the applied research of the medical science such as the scientific research in multiple fields such as developmental biology, cytobiology, metabolism, structure biology, functional genomics or tumour, senile macular degeneration SMD (AMD), nerve degenerative diseases, obesity, diabetes and pharmacy.
The present invention also provides a kind of pharmaceutical composition, it is characterized in that, the above-mentioned monoclonal antibody that contains significant quantity and pharmaceutically acceptable carrier.
The present invention also provides a kind of reagent, test kit or chip, comprises above-mentioned monoclonal antibody.
The present invention also provides the method that suppresses VEGF activity with target antibody, and use target antibody be used for the treatment of VEGF diseases related or use contain this antibody test kit carry out VEGF dependent diagnostic and detection.
Once make antibody molecule of the present invention, just can carry out purifying to it by any method of purifying immunoglobulin molecules known in the art, for example, by chromatography (for example, ion-exchange chromatography, affinity chromatography, particularly by albumin A to the affinity chromatography of specific antigens and other column chromatography), centrifugal, utilize dissolubility difference, or by the standard technique of any other protein purification.In many embodiments, antibody from emiocytosis to substratum in, by collecting substratum and carrying out purifying and obtain antibody.
Antibody can be modified by many modes, can produce other antibody or the chimeric molecule that retain original antibodies specific with DNA recombinant technology.This technology can comprise that constant region or constant region that the DNA of the immune globulin variable region of encoding antibody or complementary determining region (CDRs) is introduced to different immunoglobulin (Ig)s add framework region.Referring to, EP.A-184187, GB 2188638A or EP.A-239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of produced antibody.
Also can make by hybridoma method for monoclonal antibody of the present invention, because the DNA sequence dna of code book invention humanized antibody can be used conventional means well known to those skilled in the art, as increased and obtain according to aminoacid sequence synthetic disclosed by the invention or by PCR method, thereby also can use recombinant DNA method, available the whole bag of tricks well known in the art is connected into this sequence in suitable expression vector.Finally, be applicable to, under the condition of antibody expression of the present invention, cultivating the host cell that transforms gained, then those skilled in the art apply the conventional separation and purification means purifying of knowing and obtain monoclonal antibody of the present invention.
As mentioned above, the present invention also provides reagent, test kit or the chip for implementing antibody of the present invention.Reagent, test kit or chip at least comprise following one or more: the antibody of making according to above method, the Nucleotide of this antibody of encoding, or the eukaryotic cell that comprises this antibody, prokaryotic cell prokaryocyte and virus.Can carry out humanization by antagonist.
Other optional components of reagent, test kit or chip comprises: restriction enzyme, primer and plasmid, damping fluid etc., and for detecting the experiment of antibody activity.The nucleic acid of this reagent, test kit or chip can also have restriction enzyme site, multiple clone site, primer sites etc., is beneficial to they and being connected of non-rabbit antibody nucleic acid.Each component of this reagent, test kit or chip can separately exist in container separately, also can as required, some compatible component be assembled in single container in advance.
While preparing target antibody, can add pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to one or more organic or inorganic compositions, and it can be natural or synthetic, after combining, can promote its application with antibody.Acceptable carrier comprises that aseptic physiological saline or other are pharmaceutically obtainable and be isotonic solution and the sterile suspension of water known in the art or non-water." effective dose " refers to the dosage of the process that can improve or delay morbid state, regression or situation that damage.
Effective dose is defined on individual basis, and will be based on this, considers specifically treatment symptom and finds result.Effective dose can decide by a kind of ordinary skill of this area, and these factors that use will can not exceed normal experiment.
Biomaterial preservation explanation
Deposit number is that the cell strain of CGMCC No.3233 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 20th, 2009, and address is Datun Road, Chaoyang District, Beijing City, and Classification And Nomenclature is Chinese hamster ovary cell.
Brief description of the drawings
Fig. 1 shows that the recombinant antibodies competitive inhibition VEGF of HZD-V1, HZD-V2, HZD-V5, HZD-V6 clonal expression is combined with KDR;
Fig. 2 shows that SDS-PAGE measures the purity of HZD-V6 clonal expression EPI0030 antibody;
Swimming lane 1 is reduction electrophoresis;
Swimming lane 2 is molecular weight standard-be followed successively by from top to bottom 170kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD and 10kD;
Swimming lane 3 is non-reduced electrophoresis;
Fig. 3 is the SEC-HPLC analysis chart of the purity of HZD-V6 clonal expression EPI0030 antibody;
Fig. 4 shows the combination vigor of the direct bed board combined techniques mensuration antibody of VEGF and people VEGF;
Fig. 5 shows that EPI0030, AVASTIN suppress the IC that VEGF is combined with KDR 50measure;
The IC of EPI0030 50=166.3ng/ml, the IC of AVASTIN 50=253.7ng/ml.
Fig. 6 shows EPI0030 and the impact of AVASTIN on HCT-116 tumor growth of 5mg/kg;
Fig. 7 shows EPI0030 and the impact of AVASTIN on NCI-H460 tumor growth of 5mg/kg;
The impact of the EPI0030 that Fig. 8 shows 1.5mg/kg on NCI-H460 tumor growth.
Embodiment
Embodiment 1: hybridoma and the gene clone thereof of anti-human VEGF165 rabbit monoclonal antibody expressed in preparation
Prepare rabbit monoclonal antibodies by hybridoma cell technology.Relevant experimental program is referring to USPatent:7, and 429,487, particularly Example 1-4.
First prepare IgG Fc-hVEGF-A (Human VEGF165) fusion rotein by recombinant technology, wherein IgG Fc sequence is rabbit source.The DNA sequence dna of IgG Fc-hVEGF-A is cloned into pTT5 plasmid, and this plasmid of transient transfection enters HEK 293-6E cell strain, and serum-free culture cell is collected culture supernatant, with the IgG Fc-hVEGF-A fusion rotein of Protein A column purification transient expression.
Mix with complete Freund's adjuvant and carry out subcutaneous multi-point injection with the IgG Fc-hVEGF-A (as antigen component) of purifying, New Zealand white rabbit (New Zealand rabbits) is carried out to first immunisation, after this after mixing with purifying protein and incomplete Freund's adjuvant for every three weeks 1 time, subcutaneous injection is carried out booster immunization to rabbit, get first 4 days of spleen antigen add PBS to intravenous rabbit injection carry out final immunity.
According to the method for US Patent No. 7429487, the bone-marrow-derived lymphocyte 240E-W2 cell of the immortalization HRGTP-that rabbit splenocyte and immune spleen cell are originated identical merges in 2: 1 ratios, in 96 orifice plates with HAT culture medium culturing, then carry out filtering hybridoma, gained cell clone enters new IgG Fc-hVEGF-A in conjunction with screening.
Evaluation and screening process is divided into 2 positive colony screening steps: 1. IgG Fc-hVEGF-A antigen is immobilized onto on 96 hole Enzyme-linked Immunosorbent Assay plates, add clonal expression supernatant to hatch after 1h, with PBS washing 3 times, use the Identification of the antibodies of enzyme labelling to there is IgG Fc-hVEGF-A and clone supernatant in conjunction with active cells, thus obtain can with the positive colony of the direct combination of IgG Fc-hVEGF-A.2. by step, the positive colony in is 1. transferred to 24 orifice plates and cultivates subsequently, to obtain more expression product.IgGFc-VEGFR2 (KDR/Flk-1) extracellular region is immobilized onto on 96 hole Enzyme-linked Immunosorbent Assay plates, add IgG Fc-hVEGF-A and clonal expression product jointly to hatch 1h, again with PBS washing 3 times, use the content of the antibody test IgG Fc-hVEGF-A of enzyme labelling, with qualification clone to VEGF-VEGFR2 in conjunction with active restraining effect, thereby identify can blocking VEGF-VEGFR2 combination positive colony.
By the hybridoma cracking of screened positive colony, after extraction mRNA, reverse transcription obtains cDNA.Taking this cDNA as template, adopt PCR method to amplify respectively light chain and the variable region of heavy chain nucleotide sequence of rabbit igg antibody, to variable region of heavy chain, variable region of light chain is analyzed, the parental array (Ser Asn Asn Asp Val Met Cys Trp) that SEQ ID NO.1 is contained in the variable region of heavy chain of its coding, the parental array (Gly Cys Ile Met Thr Thr Asp Val Val Thr Glu Tyr Ala Asn Trp Ala Lys Ser) of SEQ ID N0.2 and the parental array (Arg Asp Ser Val Gly Ser Pro Leu Met Ser Phe Asp Leu Trp) of SEQ ID NO.3, the parental array (Gln Ala Ser Gln Ser Val Tyr Gly Asn Asn Glu Leu Ser) that SEQ ID NO.4 is contained in variable region of light chain, the parental array (Arg Ala Ser Thr Leu Ala Ser) of SEQ ID NO.5 and the parental array of SEQ ID NO.6 (Gly Gly Tyr Lys Ser Tyr Ser Asn Asp Gly Asn Gly).Light chain nucleic acid sequence is cloned into pTT5 plasmid.Variable region of heavy chain nucleotide sequence is cloned into the pTT5 plasmid of existing CH.Cotransfection is light, heavy chain plasmid is to HEK 293-6E cell strain, cultivates after 5 days, purifies supernatant with Protein A, finally obtains anti-human VEGF 165 monoclonal antibodies of recombinant expressed rabbit.Adopt above-mentioned positive colony screening method, the recombinant antibodies of expressing is carried out to avidity confirmation.
Embodiment 2: the preparation of humanization rabbit anti-VEGF mAb of the present invention
Humanization technology, referring to US Patent No. 7,462,697, is particularly optimized the detailed description part (DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS) of embodiment.
Adopt US Patent No. 7,462,697 described technology, use human sequence VKI-2-1-(U)-A20 JK4 and VH3-1-3-3-21 JH4 as reference sequence.The rabbit anti-VEGF mAb sequence of expressing, after humanization, respectively obtains VK and the VH of 4 versions.Variable region of light chain comprises VK-HZD1 (as SEQ ID NO.12), VK-HZD2 (as SEQ ID NO.14), VK-HZD5 (as SEQ ID NO.16) and VK-HZD6 (as SEQ ID NO.8); Variable region of heavy chain comprise with as shown in VH-HZD1 (as SEQ ID NO.11), VH-HZD2 (as SEQ ID NO.13), VH-HZD5 (as SEQ ID NO.15) and VH-HZD6 (as SEQ ID NO.7).With VK-HZD1 comparison, VK-HZD2 has 2 different residues in CDR1 district.After VK-HZD1 and 2 extra amino-acid residues of VK-HZD2N end interpolation, become respectively VK-HZD5 and VK-HZD6.VH-HZD1 is different from 71 residues of VH-HZD2, and 71 of VH-HZD1 is K, and 71 of VH-HZD2 is R.In VK (H)-HZD1 and VK (H)-HZD 2 sequences, contain rabbit source signal peptide, and contain people's source signal peptide in the sequence of VK (H)-HZD5 and VK (H)-HZD6.
The DNA sequence dna of the VK of 4 versions and VH is cloned respectively after by synthetic in the pTT5 plasmid of existing people CK sequence and people CH sequence, express antibody by people's signal peptide.By above-mentioned two plasmid co-transfection HEK 293-6E cells, transient expression humanization VEGF antibody, use the screening method in embodiment 1, the final clone who uses of HZD-V6 clone conduct that selected avidity is suitable, the humanization VEGF antibody of its expression is called EPI0030, the light chain shown in heavy chain and the SEQ ID NO.10 of its aminoacid sequence as shown in SEQ ID NO.9.
For improving output, obtain industrial production cell strain, light chain expression plasmid co-transfection shown in heavy chain by aminoacid sequence as shown in SEQ ID NO.9 and SEQ ID NO.10 enters Chinese hamster ovary cell strain (CHO), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 20th, 2009, address is Datun Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.3233.
Embodiment 3: the detection that monoclonal antibody EPI0030 of the present invention is combined with VEGF
Thereby VEGF antibody, by block the combination of itself and receptor KDR in conjunction with VEGF, suppresses VEGF signal path.After the different antibodies (HZD-V1, HZD-V2, HZD-V5, HZD-V6) that 1: 3 serial dilution is recombinant expressed and AVASTIN, (90 μ g/ml-45ng/ml) mixes with IgG Fc-hVEGF (1 μ g/ml), add the antibody-VEGF mixture mixing to spreading in the hole of IgG Fc-VEGFR2 plate, add after mouse-anti people VEGF antibody and detect with sheep anti-mouse igg antibody-AP colour developing.Result shows the activity that the expressed recombinant antibodies competitive inhibition VEGF similar with AVASTIN is combined with KDR.The antibody that measurement result shows HZD-V1, HZD-V2, HZD-V5, HZD-V6 clonal expression all can blocking VEGF and the combination (seeing Fig. 1) of KDR.
With reference to above-mentioned experimental technique, measure the IC of EPI0030 and AVASTIN 50value is half-inhibition concentration, and result as shown in Figure 5, shows that EPI0030 and AVASTIN have similar competition to suppress vigor.
Adopt BIAcore-3000 to measure the dissociation constant Kd of EPI0030 and VEGF.People VEGF is fixed on CM5 chip, and the EPI0030 of 2 times of serial dilutions and AVASTIN are injected in the HBS-EP damping fluid that flow velocity is 30ul/min.Kd is k off/ k on, in table 1, showing the dissociation constant of EPI0030 and AVASTIN and people VEGF, EPI0030 and VEGF dissociation constant are 0.485nM, are 1/100 of AVASTIN, illustrate that the binding ability of EPI0030 and VEGF is better than AVASTIN.
The dissociation constant of table 1.EPI0030 and AVASTIN and people VEGF
Figure GPA00001338091300171
Embodiment 4: the qualification of monoclonal antibody EPI0030 of the present invention and the mensuration of external activity
The EPI0030 antibody of HEK 293-6E cell HZD-V6 clone transient expression, after purifying, has carried out the calibrating about quality, and concrete certified variety is as follows:
A: purity
Adopt SDS-PAGE (reduction and non-reduced) and SEC-HPLC to carry out purity assay, the results are shown in Figure 2, Fig. 3, the purity that shows EPI0030 antibody is greater than 98%, Content of polymer is less than 5%, main peak (retention time 10.572 minutes) area accounts for the total area (comprising peak 1,2,3 from left to right) and is greater than 95%, and peak 1,2 is polymer.
B: external combination activity
IgG Fc-hVEGF bed board, 1%BSA sealing, 1: 3 serial dilution EPI0030 antibody and 8 gradients of AVASTIN (1 μ g/ml-0.46ng/ml), be added in the hole of completing IgG Fc-hVEGF, adds the anti-human IgG antibody of donkey-AP and detect.Result shows that EPI0030 antibody has similar combination vigor with AVASTIN.
Adopt ELISA method, comprise the direct bed board method of IgG Fc-hVEGF (seeing embodiment 1) and KDR competition combined techniques (seeing embodiment 3), result shows that the combination of EPI0030 antibody and VEGF and the combination of inhibition VEGF and KDR are dosage correlation, the results are shown in Figure 4 and Fig. 5.
Embodiment 5: the activity in vivo of monoclonal antibody EPI0030 of the present invention detects
From liquid nitrogen container, take out a frozen human colon carcinoma HCT-116 cell and Non-small cell lung carcinoma NCI-H460 cell (approximately 1 × 10 at experiment first two weeks 7cell) put rapidly to 37 DEG C of water-baths and melt, with being preheated to 37 DEG C of McCoy's 5A substratum and DMEM substratum that added 10% foetal calf serum (purchased from GIBCO), cell is seeded to 75CM respectively subsequently 2tissue Culture Flask in, in the time of Growth of Cells to 80% fusion rate, went down to posterity by 1: 5, continuous passage is more than three times in vitro.When cell total amount reach inoculation required, by trysinization for cell, centrifugal, then remove serum, the human colon carcinoma HCT-116 finally handling well with serum-free, antibiotic-free McCoy's 5A substratum and the adjustment of DMEM substratum respectively and Non-small cell lung carcinoma NCI-H460 cell density to 5 × 10 with PBS washed cell 7/ ml, cell suspension is positioned on ice, is inoculated in nude mice veutro portion in age in 6-8 week, every mouse inoculation 0.1ml, 5 × 10 6cell/only.Within every 2 days, measure the diameter of tumor of nude mouse with vernier callipers, treat that the tumour of every mouse is all grown to 100mm 3-300mm 3, be divided at random 5 groups, 6 every group after entering group by knurl volume SD < 1/3.Human colon carcinoma HCT-116 model group is respectively model control group (1 group), 5mg/kg AVASTIN (1 group) and 5mg/kg EPI-0030 (1 group) group, by AVASTIN and EPI-0030 with normal saline dilution to 0.5mg/ml.Non-small cell lung carcinoma NCI-H460 model group is respectively model control group (1 group), 5mg/kg AVASTIN (1 group) and 1.5mg/kg, 5mg/kgEPI-0030 (2 groups) group by AVASTIN and EPI-0030 normal saline dilution to 0.5mg/ml and 0.15mg/ml.Administration group respectively weekly 1,3,5d gives respectively AVASTIN and the EPI-0030 of disposable celiac injection 0.2ml, and model control group gives physiological saline 0.2ml with same time and mode, successive administration 3 weeks (21 days, totally 9 times).
Measure tumour knurl tubercle maximum diameter a and the path b of nude mice, by formula V=0.5 × a × b 2calculate gross tumor volume, using relative tumor proliferation rate T/C% as index of assessment of curative effect.
Result demonstration, in human colon carcinoma HCT-116 model, in 5mg/kg dosage group, the tumour inhibiting rate of EPI-0030 and AVASTIN is respectively 65.7% and 15.7% (seeing Fig. 6).In Non-small cell lung carcinoma NCI-H460 model, the tumour inhibiting rate of EPI-0030 and AVASTIN is respectively 95.5% and 66.7% (seeing Fig. 7); In 1.5mg/kg dosage group, the tumour inhibiting rate of EPI-0030 is 57.6% (seeing Fig. 8).Presentation of results EPI-0030 has the effect of significant inhibition tumor promotion.
With reference to AVASTIN clinical indication, select human colon carcinoma HCT-116 and the restraining effect of Non-small cell lung carcinoma NCI-H460 bare mouse different species transplanted tumor modelling verification EPI-0030 to human colon carcinoma and Non-small cell lung carcinoma, prove that EPI-0030 has the effect of remarkable inhibition human colon carcinoma and Non-small cell lung carcinoma xenograft tumor.EPI-0030 antitumor activity in model is better than AVASTIN.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (12)

1. one kind has the active monoclonal antibody of specific binding VEGF, it is characterized in that, the aminoacid sequence shown in SEQ ID NO.1, SEQ ID N0.2 and SEQ ID NO.3 is contained in its variable region of heavy chain, and the aminoacid sequence shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 is contained in its variable region of light chain.
2. monoclonal antibody according to claim 1, is characterized in that, described monoclonal antibody comprises single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody.
3. monoclonal antibody according to claim 1 and 2, is characterized in that, its weight chain variable region amino acid sequence is as shown in SEQ ID NO.7.
4. monoclonal antibody according to claim 1 and 2, is characterized in that, the aminoacid sequence of its variable region of light chain as shown in SEQ ID NO.8.
5. monoclonal antibody according to claim 1 and 2, is characterized in that, the aminoacid sequence of its heavy chain as shown in SEQ ID NO.9.
6. monoclonal antibody according to claim 1 and 2, is characterized in that, its light-chain amino acid sequence is as shown in SEQ ID NO.10.
7. according to the monoclonal antibody described in claim 1-6 any one, it is characterized in that, heavy chain amino acid sequence is as shown in SEQ ID NO.9, and light-chain amino acid sequence is as shown in SEQ ID NO.10.
8. a cell strain, is characterized in that, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.3233.
9. the monoclonal antibody described in claim 1 to 7 any one is in the purposes for the preparation of in the diseases related medicine for the treatment of VEGF.
10. purposes according to claim 9, is characterized in that, diseases related tumour, senile macular degeneration SMD, nerve degenerative diseases, obesity, the diabetes of comprising of described VEGF.
11. 1 kinds of pharmaceutical compositions, is characterized in that, monoclonal antibody and pharmaceutically acceptable carrier described in the claim 1-7 any one that contains significant quantity.
12. 1 kinds of reagent, test kit or chips, comprise the monoclonal antibody described in claim 1 to 7 any one.
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